Purification of antibiotics



Patented Dec. 29, 1953 assent UNITED STATES PATENT OFFICE 2,664,419, PURIFICATION or ANTIBIOTICS No Drawing. Original an Serial No. 767,851.

tion December 8, 195

15 Claims.

This application is a division of our application Serial No. 767,851, filed August 9, 1947, now Patent No. 2,537,933, dated January 9, 1951.

This invention relates to basic antibiotics of the streptomycin type, i. e., to members of the genus composed of streptomycin and antibiotically-active bssic compounds which (like streptomycin) are capable of forming water-soluble salts with acids such as sulfuric and water-insoluble salts with organic-base-precipitating reagents (e. g., derivatives of streptomycin, such as dihydrostreptomycin. and similarly-acting antibiotics, such as streptothricin) In 1944, Schatz, Bugie, and Waksman (Proo. Soc. Exp. Biol. Med, 1944, 57, 244) showed that a potent antibiotic, termed streptomycin, was formed during the gro th of the organism Actz'nomy/ce crz'eeu (now called Streptomyces grz' eus) and this antib otic has s nce been demonstrated to be of high clinical utility.

It was liter found that a number of streptomycins are formed at the same time. The first streptomycin obtained in pure crystalline form (as a reineckate) and fully characterized (Wintersteiner and Fried application, Serial No, 656.541, filed May 1, 1946. now Patent No. 2,501,014, dated March 21, 195 is now referred to rs streptomycin A: and the second streptomycin characterized (Fried and Titus application Serial No. 737,400, filed March 26, 194 now Patent No. 2,565,653, dated August 28. 1951) is now reerred to as streptomycin B. Moreover, there are indications that still other streptomycins are formed at the same time and/or may be formed at the same time by ch: nges in the culture conditions; and it is intended that each of such antibiotics and any mixtures thereof (whether in the form of the free base or water soluble salt thereof) be comprehended bythe term streptomycin when employed unmodified hereinafter.

Streptomycin has been purified heretofore by various methods, all of which were complex plication August 9, 1947, Divided and this applica- 0, Serial No. 199,921

and/or inefiicient (and consequently expensive). For example, a method widely employed prior to this invention essentially comprised the following steps: (1) trecting a primary streptomycin-containing liquid with an activated charcoal, which selectively adsorbs the streptomycin;

(2) eluting the streptomycin from the charcoal with an aqueous, water-soluble mineral acid, preferably at a slightly elevated temperature (say about 30-50 C.) (3) treating the eluate with an organic-base-precipitating reagent, preferably phosphotungstic acid; and (4) decomposing the precipitate. [The term primary streptomycincontaining liquid comprehends, inter zlia: (a) the culture liquid obtained by growing Streptomi/C68 grz'seus under conditions and in a medium suitable for the production of streptomycin, and removing the solids from the the medium; (b) the culture liquid of enhanced potency obtained by acidification of such culture (with hydrochloric or sulfuric acid, for example), the liquid being neutrclized; and (c) the liquid obtained by acid-extraction or the solids separated from such culture, the liouid being neutralized] It is the object of this invention to provide simple, efiicient, and otherwise advantageous methods of purifying ba ic antibiotics of the streptomycin type-especially streptomycin; and it is a further object of this invention to provide methods of preparing certain salt-type derivatives of basic antibiotics of the streptomycin type.

It has been found that basic antibiotics of th streptomycin type interact with surface-active agents of the organically-substituted polvbasicinorganic-acid type to form certain salt-type combinations which are much less soluble in water and more soluble in certain water immiscible organic solvents then the antibiotics; and it has also been found that such salt-"type derivatives of the antibiotics may be decomposed to recover the antibiotics.

The methods of this invention essentially comprise intimately contacting an aqueous solution of a basic antibiotic of the streptomycin type (especially streptomycin) with a surface-active agent of the organically-substituted polybasicinorganic-acid type and a substantially waterimmiscible organic solvent for soaps (including invert soaps), preferably with a substantially water-immiscible aliphrtic alcohol, and notably with amyl alcohol mixtures; and the purification methods of this invention essentially comprise intimately contacting an aqueous solution of an impure basic antibiotic of the streptomycin in type (e. g., a primary streptomycin-containing liquid, or an aqueous solution of a pr rtially-purified streptomycin, such as the eluate referred to hereinbefore) with a surface-active agent of the organically-substituted polybasic-inorganic-acid type and a substantially water-immiscible or anic solvent for soaps, recovering the orgrnic solvent phase, and converting the salt-type derivative of the antibiotic therein into a water-soluble salt of the antibiotic, preferably by intimately contacting the organic solvent solution with an aqueous, water-soluble, relatively-strong acid (especially with an aqueous, water-soluble, relatively-strong mineral acid), and recovering the aqueous phase. The water-soluble salt (of the basic antibiotic of the streptomycin type) recovered from the aqueous solution thus obtained is considerably purer than the antibiotic treated, and the recovery of antibiotic ectivity in the purification treatment is of a high order. By the practice of this invention, it is possibl to obtain uniformly high yields of relatively-pure streptomycin (for example) having a potency above about 400 units/mg.

Among the preferred surface-active agents of the organically-substituted polybasic-inorganicacid type for the purposes of this invention are those of the formula R-OX-O-Y wherein R is the residue of a substantially water-immiscible organic hydroxyl compound, --O-X-O- is the divalent acid-residue of a Water-soluble polybasic inorganic acid (e. g., sulfuric or phosphoric acid),

and Y is a member of the group consisting of H and cations forming water-soluble salts with the anion RO-X-O. Notable among such wetting agents for the purposes of this invention are those in which the polybasic-inorganic acid is sulfuric, and the acid is partially esterified with a higher aliphatic alcohol, i. e., wetting agents of the group consisting of mono (higher-ali hatic) esters of sulfuric acid, and water-soluble salts thereof.

' Other preferred surface-active agents of the organically-substituted polybasic-inorganic-acid type for the purposes of this invention are those of the group consisting of aromatic sulfonic acids, sulfonated oils, sulfonated higher fatty acid derivatives, and water-soluble salts thereof.

Among the partial higher-alkyl esters of sulfuric acid utilizable in the practice of this invention are: the group of sodium salts of sulfates of synthetic higher aliphatic alcohols, such as CeI-IeCI-I (021-15) CzI-LrCI-I SOiNEt) C2H4CH(C2H5) 2 [e. g., Tergitol Penetrant 7],

canon c nsioznlcn soina CH2CH(CH3) 2 [e. g., Tergitol Penetrant G8]; and the series of partial sulfuric-acid esters of higher aliphatic alcohols and their salts, such as sodium octyl sulfate, sodium oieyl sulfate, sodium cetyl sulfate, sodium stearyl sulfate, and sodium lauryl sulfate [e. g., Aurinol, Wetanol, the Duponols, and the Gardinolsl. Among the aromatic sulfonic acids. sulfonated oils, and sulfonated higher fatty acid derivatives utilizable in the practics of this invention are: the sodium sulfonates of higher fatty acid esters and amides, such as the sodium salt of sulfonated ethyl (or other alkyl) oleate [e. Igepon AP Extra], and CmHaaCONHCzI-hSOzNa [e. g., Igepon T]; the sodium sulfonates of petrov leum hydrocarbons [e. g., Ultrawet]; the sodium salt of a polyal {Y1 benzene sulfonic acid having ten carbon atoms [e. g, Ultrawet, 40A], and other sodium alkyl aryl sulfonates [e. g., Nacconol NRSF]; and Turkey red oil (i. e., sulfonated castor oil) Among the partial esters of phosphoric acids utilizable in the practice of this invention are dicresyl phosphate, lecithin, and a phosphorated higher alcohol of the formula (capryl) sNasi-eozo [e. e., W. A. 58].

The substantially water-immiscible organic solvents for soaps utilizable in the practice of this invention include, inter alia: aliphatic alcohols, such as n-amy1 (l-pentanol), n-butanol (1- butanol), sec. butanol, methyl-isobutyl-carbinol, methyl-amyl-carbinol, methyl-isopropyl-carbinol, isobutyl-carbinol, Z-ethyl-hexanol, and amyl alcohol mixtures, such as refined fermentationamyl-alcohol; aliphatic alcohol esters of lower fatty acids, such as the acetate of methyl-isobutyl-carbinol, and amyl acetate; aliphatic ketones, such as methyl-isobutyl ketone; aliphatic others, such as di-n-butyl ether, and diethyl ether; hydrocarbons, such as benzene and toluene; and halogenated hydrocarbons, such as ethylene dichloride, chloroform, and carbon tetrchloride.

Among the water-soluble, relatively-strong acids utilizable for recovery of the antibiotics from their salt-type combinations with the surface-active agents are sulfuric, hydrochloric. phosphoric, oxalic, citric, sulfamic, and nitric.

For maximum efficiency, the amount of the surface-active agent employed should be in excess of that required to combine with all the antibiotic in the solution treated, the optimum amount of surface-active agent being therefore dependent on the concentration of the antibiotic solution treated and the potency of the antibiotic.

In one embodiment of the invention, an aqueous solution of an impure antibiotic of the streptomycin type i intimately contacted with a solution of the surface-active agent in a substantially water-immiscible organic solvent for soaps; the organic solvent phase is recovered and intimately contacted with an aqueous, watersolu ble, relatively-strong acid; and the aqueous phase is recovered and dried, preferably freeze-dried (i. e., frozen and subjected to a high vacuum to sublime off the water). Alternatively, the intimate contact between the antibiotic solution, surface-active agent, and organic solvent may be effected by first mixing the antibiotic solution with the surface-active agent, and then in timately contacting the mixture with the organic solvent; or by first mixing the antibiotic solumum transference of streptomycin from the aqueous solution treated is obtained when the pH of the aqueous phase is adjusted to between about 2 and about 8, especially between about 3 and 4. j

Alternatively, the salt-type combination of the antibiotic and the surface-active agent maybe recovered from the. solution in the'organic solvent and then converted into a water-soluble salt, or

used as a therapeutic agent per se. the

salt-type derivative of the antibiotic may be recovered by evaporating oh the organic solvent in vacuo, or (where the organic solvent lends itself to such procedure, as in the case of benzene, for example, by freeze-drying the solution). Alternatively, the salt-type derivative of the antibiotic may be obtained more directly by the precipitation method described and claimed in application Serial No. 767,852, file-d August 9, 1947, now Patent No. 2,537,934, dated January 9, 1951. These salt-type derivatives of antibiotics are, in general, oil-soluble or oil-dispersible; and they may be used therapeutically, e. g., orally administered either per se or in oily media for the treatment of intestinal disorders (relying on intestinal processes to liberate theantibiotic in water-soluble form), or parenterally administered in oily media (or administered) by implantation of a pellet of the solid) for prolonged antibiotic action. Where production of the salt-type derivative of the antibioticrather than purification of the antibioticis the objective, one may employ a reconstituted aqueous solution of the antibiotic (e. g., an aqueous solution of the highly purified or pure. antibiotic).

The various extractions involved in the methods of this invention may of course be carried out by the countercurrent technique; and the spent solutions and/or extracts may be reemployed in the method for further removal ofactivity and/or concentration of the extracted material. Thus, the spent antibiotic-containing liquid may be extracted with a fresh batch of surface-active agent and organic solvent; the spent organic solvent may be extracted with a fresh batch of aqueous, water-soluble relativelystrong acid, or such solvent maybe used on a fresh batch of antibiotic-containing liquid; and/or the aqueous acid extract may be used to treat a fresh batch of organic solvent solution, to build up the antibiotic concentration therein.

The following examples are illustrative of the invention (all solutions or dilutions referred to without identification of the solvent or diluent being solutions in or dilutions with water):

Example 1 v ((1)1350 mg. streptomycin.hydrochloride is dissolved in '70 ml. water saturated with n-butanol; the, solution is mixed with 70 ml. of a 7% solution of sulfated oleyl alcohol (e. g., Aurinol DS) in n-butanol saturated with water; the mixture is shaken in a separatory funnel for 5 minutes at room temperature; and the n-butanol layer formed on standing is recovered. About 94.9% of the streptomycin in the aqueous solution is thus extracted into the n-butanol (as a salt-type combination of streptomycin and the sulfated oleyl alcohol).

Similarly, about 90-96% of the streptomycin present in aqueous solutions in concentrations of AGO-4,000 units/mL- maybe extracted into l5- acid e ters or h her alipha ic alcqh ls (e.

n-butanol with the aid of other partial sulfuric- Duponol-C or Turkey red oil) on similar extraction of a streptomycin-containing culture filtrate [obtained by growing Streptomyces griseus in submerged culture in an aqueous medium containing soybean meal, dextrose, and. sodium chloride, acidifying the incubated culture, and filtering] having a potency of 391 units/mL, and a pH of about 5-7, preferably about 6.1, about -95% of the culture-filtrate acti ity is extracted into the n-butanol; and on similar extraction of a streptomycin-containing eluate [obtained by treating a streptomycin-containing culture filtrate with an activated charcoal, and eluting the streptomycin from the charcoal with dilute hydrochloric, nitric, or sulfuric acid] having a potency of 201 units/ml. and a pH of 6.8, about -98% of the eluate activity i extracted into the n-butanol.

(b) 48 m1. of the n-butanol solution of the salttype combination of streptomycin and the sulfated oleyl alcohol is shaken with 48 ml. water in a separatory funnel at room temperature; and 5% hydrochloric acid is added dropwise with shaking until the pH reaches 2.0. The separated clear aqueous phase is passed through a column o'f anion-exchange-resin (e. g., Amberlite IR-4B; cf. U. S. Patent 2,402,384, dated June 18, 1946), to bring the pH up to 7.6, and then freeze-dried. The streptomycin hydrochloride thus obtained, in a. yield of about 53%, has a potency of about 542 units/mg.

The salt-type combinations of streptomycin and other partial sulfuric-acid esters of higher aliphatic alcohols or Turkey red oil obtained as described in section (a) of this example may be similarly decomposed.

(2'): Alternative) 80 ml. of the n-butanol solution of the salt-type combination of streptomycin and the sulfated oleyl alcohol is cooled, passed through a 5-inch. (32 g.) column of sulfuricacid-washed alumina (pl-I 4.0) under suction; and the column is eluted with a 0.1 normal solution of hydrochloric acid in anhydrous methanol. The eluate is collected in 10 ml. fractions; and the active fractions (or fractions 3 to 9 inclusive) are combined and treated with anhydrous ether to precipitate the streptomycin hydrochloride. The precipitate is separated by centrifugation, dissolved in a minimum quantity of dry methanol, and reprecipitated with dry ether. The precipitate (separated by centrifugation) is dissolved in about 5 ml. water, and the solution is freeze-dried. The streptomycin hydrochloride obtained (in an over-all yield of about 56%) has --a-p0tency ofabout 636 units/mg.

Similar decomposition of the salt-type combinations obtained from the following streptomycins and surface-active agents give strepto- Freeze-dried Salt-type combination oi streptomycin hydrochloride Streptomycin Surface-active agent Potency veldv units/mg. percent 92 units/mg Partial sulfuric-acid 470 78 ester of hiEher aliphatic alcohol (e. g., Duponol tion, while stirring; and the mixture is stirred for an hour, and the amyl alcohol layer is recovered.

(b) The amyl alcohol extract is extracted 3 times with '75 ml. portions of aqueous sulfuric acid of pH 0.5; the acid extracts are combined and treated with barium hydroxide solution to pH 8-10; and the precipitate formed is filtered off, The filtrate is adjusted to pH 6.2 with sulfuric acid; the precipitated barium sulfate is filtered off and the filtrate is freeze-dried, yielding about 1.0 g. streptomycin sulfate having a potency of about i133 units/mg.

For brevity, the details of other examples pro- 5 l l r l iifiml 391 239 3 cedurally analogous to Example 3 but illustrating e, Turkeyred oil 105 I 38 variations of the invention are given hereinafter in tabular form (Table I) TABLE I H a P H r p a llllC 1101 n O pH ad- Type of streptomycin justed extract filtrate ig igg Example Surface-active a ent Organic solvent used, ggg gg gf ia i gig fiig and potency used and quantity and qummty furic acid treated, quantity, hydroxide ium hyaqueous g'?' and potency solution droxide sulfuric e tosolution acid to- 0 4 solution of Tergitol Refinediermentation- 3.7 Eluate, 8 liters, 421 3.4 11.5 6.0 3.5 g., 443

Penetrant 7 (ct. Examyl-alcohol,1l1ter. units/ml. units/mg. ample 2) 250 ml.

5 Saturated aqueous solu- Refinedfermentation- 3A Eluatc, 4 liters, 462 3.5 11.3 6.0 3g.,483units/ tion of Ultrawet, 40A amyl-alcohol, 500 ml. units/ml.

(05. Example 3), 134 m.

5 Saturated aqueous solulllethyl-amyl-earbinol, 3.2 Elnate, 8 liters, 337 3.4 12.5 6.4 3.5 g.. 200

tion of Ultrawet, 40A 1 liter. urnts/ml. units/mg (05. Example 3), 250 m.

7 do Amylaoetatc, llitern 3.4 Eluate, 8 iters, an 3.65 12 6.2 6g.,408unitsl units/ml. mg.

8 Saturated aqueous solu- Refinedfermentation- 3.4 Culture-filtrate, 8 li- 3.5 0,6 8a., units/ tion of Ultrawet, A amyl-aleol1ol,ll1ter. ters, 180 units/ml. mg

(of. Example 3), 300 ml.

Example 2 Example 9 (a) 170 ml. of a 25% aqueous solution of the surface-active agent [e. g., Tergitol Penetrant ll is added to 4 liters of a streptomycin-containing culture filtrate having a potency of 142 units/ml.; the mixture is adjusted to pH 6.35 with 10% sodium hydroxide solution, while stirring; and 670 ml. of refined fermentation-amyl-alcohol is added, and the mixture is stirred for 5 minutes. The amyl alcohol phase, separated by centrifuging, contains about 85% of the activity of the culture filtrate.

(b) 250 ml. Water is added to the amyl alcohol solution, and the mixture is adjusted to pH 0.5 by adding 10% sulfuric acid while stirring. The aqueous extract formed on separation of the layers is stirred with an anion-exchange resin of the type described hereinbefore (e. g., Amberlite lR- i-B) to decrease the acidity to pH 5.7. The aqueous extract, which contains about 60% of the activity of the culture filtrate, is freezedried, yielding about 7.1 g. streptomycin sulfate having a potency of about 53 units/mg.

sample 3 (a) 500 ml. of a saturated aqueous solution of an alkylated monosodium benzene sulfonate having several allsyl groups totaling ten carbon atoms (e. g., Ultrawet, 40A) is mixed with 300 ml. refined fermentation-arnyl-alcohol, and the mixture is adjusted to pl-l 2.0 by adding sulfuric acid while stirring. The amyl alcohol layer is separated and added to 3 liters of a streptomycinhydrochloride-containing eluate having a potency of 296 units/ml; the mixture is adjusted to pH 3.3 by adding aqueous sodium hydroxide solu- (a) 3.5 liters of a streptomycin-hydrochloride eluate (280 units/mg.) is adjusted to pH 5.0 with sodium hydroxide solution, and 40 g. sulfonated castor oil and 500 ml. amyl acetate are added thereto. The mixture is stirred for an hour; and the amyl acetate layer is separated.

(b) The amyl acetate solution is extracted with N/lO sulfuric acid; the aqueous layer is separated, neutralized with barium hydroxide solotion, and filtered; and the filtrate i freezedried, yielding about 0.3 g. streptomycin sulfate (potency about 337 units/mg).

Example 10 (a) 4 liters of a streptomycin-hydrochloride eluate (185 units/ml.) is adjusted to pH 7.0 with sodium hydroxide solution, and the precipitate formed is filtered off. 20 g. solid sodium salt of polyalkyl benzene sulfonate (e. g., Ultrawet E) is added to the filtrate, the pH is adjusted to 3.5 with dilute sulfuric acid, one liter refined fern-zen tation-amyl-alcohol is added, the mixture is stirred for an hour, and the amyl alcohol layer is recovered.

(1)) The amyl alcohol solution is extracted, a times with 50 m1. portions of dilut sulfuric acid (pl-l 0.5); and the acid extracts are combined, and adjusted to pl-I 12.0 with barium hydroxide. The resulting precipitate is filtered on, the filtrate is adjusted to pH 5.9 with dilute sulfuric acid, and filtered; and the filtrate is freeze-dried, yielding about 2 g. streptomycin sulfate (potency about 291 units/mg).

For brevity, the details of other examples procedurally analogous to Example 10 but illustrating'iurther variations of the invention are given hereinafter in tabular form (Table II).

tol Penetrant 4 (of. Example 16) and 500 ml. reefined fermentation-amyl-alcohol are added to ,3

Table II aci Tyne of sttrepto- H d S f f O I t O I t extaact figggxlglladte mycm-con ainmg p a ur ace-ac lve rganic so ven rgamc so ven a g g ol ti n'treated, justed g fi agent'added, added,-and layer extracted justed gg fig quantity. and toandq v qu n ty ht a gr 'potency barium obtamgd hyroxidc to 11 Sulfuric-acid 6.7 Powdered bari- Ult-rawet-E (cf. Reilncdfermenta- Dilute sulfuric 12 lg.,396 units/ eluate, iliters, uIn hydroxlde. Example 10' tion-amyl-alcoacid, p11 3.0. mg. l40'units/ml. g. 1101, 500 ml.

12 ,Eluate,4.5liters, Neutral- Sodium hydrox: Ultrawet-E (cf. Amyl acetate, 300 Dilute sulfuric 12 lg.,3l9units/ 140 units/ml. ity. ,xde solution. Example l0), mL, 300 ,ml. acid, pH 0.5, 50 mg g. extnaetlons). -ml., 50 ml. (2 extractions).

13 Sulfuric-acid .Amberhte .IR- .Reilned e a- No ma suliuric 7 1.63 g. ,417

eluate, 10 li- 6.0 4B (cfpExain- Ultrawet-E (of; tlOH-EITlYl-dlCO acid. units/mg. ters,140units/ e Example 10), hol, 1v liter.

ml. 7.3 Begum hydrox' l4 Eluate, 4 liters, 8 Ammonium hy- 4.6% solution of Refinedfermenta- Sulfuric acid or 12 1.28 g. 252

250 it l, drox de soluthe acid form of tion-ainyl-llcopH 3.7, sulfuric units/mg.

tion. Tergltol Pcnehol, 200 ml. acid of pH 0.5.

trant 7 (cf. Ex-- ample 2) in re-, fined fermentation-amyl-alcohol, 190 ml.

Example 15 liters of a streptomycin-containing culture fil 1 g. streptomycin having a potency of 2,28 units/mg. is dissolved in 1 liter water, and 25 ml. of a solution .of Tergitol Penetrant '7 (cf. Example 2) is added, followed by 100 ml. refined fermentation-amyl-alcohol; the mixture is adjusted to pH 3.3 whilestirring, and stirred for an additional half-hour; and the amyl alcohol layer formed is recovered. 'The amyl alcohol solution, containing about 75% of the streptomycin in the aqueous solution treated, is extracted with dilute sulfuric acid (pH 0.5) and the aqueous acid ex tract, containing about 80% of the streptomycin in theamyl alcohol solution, is further treated as describedhereinbefore.

Example 1.6

(a) 150 m1. of a 25% aqueous solution of the surface-active agent C4H9CH (Cal-I5) CzHiCH SOiN a) CH2CH(-CH3)2 ie. g.,Tergito1 Penetrant 4] and 500 ml. refined fermentation-amyl-alcohol are added to 3 liters of a streptomycin-containing culture filtrate (potency 50 units/m1; pH 1.5); the is adjusted to 4.0 and the mixture is shaken; the resulting emulsion is broken by centrifuging; and the amyl-alcohol layer (containing about 95% of the culture filtrate activity) is recovered.

'(b) The amyl alcohol solution is extracted with 25% sulfuric acid to pH 0.5; the aqueous layer formed is recovered, neutralized to pH 6.8 with barium hydroxide, and filtered; and the filtratev is freeze-dried, yielding a white solid, representing about 97% of the activity of the amyl alcohol solution.

Modification of the procedure detailed in (a) to the extent of treating a streptomycin-containing culture filtrate having a I potency of 115 units/ml. and a pH of 1.7, using 200 m1. of a 25% solution of a higher aliphatic alcohol sulfate (e. g., a Duponol) in place of the'Tergitol Penetrant l, and adjusting the pH to 3.5 instead of 4.0, yields an amyl alcohol solution containing about 90% of the activity of the culture filtrate.

Example 1,? (a) 150ml. of a 25% aqueoussolution of Tergltrate ,(potency units/ml; pH 1.6) the pH is adjusted to'3.5 and the mixture is further treat.- ed asdescribed in sections (a) and (b) of Example 1 6 to obtain a spent amyl alcohol layer and an aqueous acid extract. [The spent amyl alcohol is then used With 50 ml. additional solution of Tergitol 'Penetrant 4 to extract 3 liters of new streptomycin containing culture filtrate (and the spent amylalcohol from this extraction may in turn be used to extract new culture filtrate in the same way) 'Ihe aqueous acid extract is treated as further described in section (b) of Example 16. Alternatively, the aqueous acid extract is then used to extract the amyl alcohol extract ob,- tained from a new batch of streptomycin-containing culture filtrate, thus building up the concentration of streptomycin sulfate in the aqueous acid extract.

, Example v18 (at) .100 ml. of an aqueous solution of streptomycin hydrochloride having. a potency of 10,400 units/m1. is intimately mixed with 50 ml. ether containing 11.3 ml. ,Tergitol Penetrant '7 (25% aqueous solution). After standing, the ether layer formed (containing; about 91% of the activlil of the aqueous, solution treated) is recovered.

Substitution of benzene for the ether results in extraction of about 98% of theactivity. 'l'he emuls1iicat-0n vo'liinculties encountered with this solvent are overcome by allowing the mixture to stand for 1 -16 hours in he cold before separating the layers.

(b) .An etner solutionobtained as described in section (a) containing streptomycin derivative equivalent to 1,016,0110 units streptomycin, is mixed with asolution of ,5 g. A1013 in 25 ml. ether; the precipitate formed is removed by centrifugation, and dissolved in- Water; the aqueous solution is neutralized with diluted potassium hydroxide solution; and the solution is filtered and freeze-dried, yielding about 3.62 g.-streptomycin hydrochloride having a potency of 222 units/mg. (ayieldof about 79%).

In place of the A1013 used for the decomposition of .theesalt-type'derivative of streptomycin, one

11 may employ equivalent salts, such as ZnClz or FeCls.

Among other basic antibiotics of the streptomycin type which may be treated by the methods of this invention are streptothricin and dihydrostreptomycin; and among other basic antibiotics of the streptomycin type utilizable for the preparation of salt-type combinations with the surface-active agents in accordance with this invention are pure (or substantially-pure) streptomycin A, streptomycin B, dihydrostreptomycin A, and dihydrostreptomycin B.

The basic antibiotic of the streptomycin type purified by the method of this invention may be further purified by repetition of the same purification method. Also, it may be preliminarily purified or further purified by any other method, especially by one of the following: (I) intimately contacting an aqueous solution of the antibiotic with a substantially water-insoluble carboxylic acid and a substantially water-immiscible organic solvent for the carb-oxylic acid, recovering the organic solvent phase, and converting the salt-type derivative of the antibiotic therein into a water-soluble salt of the antibiotic; (cf. application Serial No. 762,205, filed July 19, 1947, now Patent No. 2,644,816, dated July 7, 1953); (II) treating an aqueous solution of the antibiotic with a water-soluble salt of a substantially water-insoluble carboxylic acid, recovering the precipitated salt-type combination of the antibiotic and the carboxylic acid, and converting it into a water-soluble salt of the antibiotic of. application Serial N0. 762,206, filed July 19, 1947, now Patent No. 2,631,143, dated March 10, 1953); and (III) intimately contacting an aqueous solution of the antibiotic with a surface-active agent of the organically substituted polybasic inorganic-acid type recovering the precipitated salt-type combination of the antibiotic and the surface-active agent, and converting it into a water-soluble salt of the antibiotic (cf. Patent No. 2,537,934).

The invention may be variously otherwise embodied within the scope of the appended claims.

We claim:

1. The method which comprises. intimately contacting an aqueous solution of an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, and streptothricin with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkylaryl sulfonates, and sulfonated castor oil.

2. The method which comprises intimately contacting an aqueous solution of streptomycin with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil.

3. The method which comprises intimately contracting an aqueous solution of an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, and streptothricin with a substantially water-immiscible aliphatic alcohol and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil.

4. The method which comprises intimately contacting an aqueous solution of an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, and streptothricin with a refined fermentation-amyl-alcohol and a watersoluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil.

5. The method which comprises intimately contacting an aqueous solution of an antibiotic of the group consisting of streptomycin, dihydihydrostreptomycin, and streptothricin with a substantially water-immiscible organic solvent for soaps and water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkylaryl sulfonates, and sulfonated castor oil, separating the organic solvent phase, and recovering the salt-type combination of the antibiotic and the surface-active agent from its solution in the organic solvent.

6. The method of purifying an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, and streptothricin which comprises intimately contacting an aqueous solution of the impure antibiotic with a substantially waterimmiscible organic water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, recovering the organic solvent phase, and

converting the salt-type derivative of the antibiotic therein into a water-soluble salt of the antibiotic.

7. The method of purifying streptomycin, which comprises intimately contacting an aqueous solution of an impure streptomycin with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, recovering the organic solvent phase, and converting the salt-type derivative of streptomycin therein into a water-soluble salt of streptomycin. W

8. The method of purifying streptomycin, which comprises intimately contacting an aqueous solution of an impure streptomycin with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulionates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, recovering the organic solvent phase, intimately contacting the organic solvent solution with an aqueous, water-soluble, relatively-strong acid, and recovering the aqueous phase.

9. The method of purifying streptomycin, which comprises intimately contacting a primary streptomycin-containing liquid with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, recovering the organic solvent phase, intimately contacting the organic solvent solution with an aqueous, watersoluble, relatively-strong acid, aqueous phase.

10. The method of purifying streptomycin, which comprises treating a primary streptomycin-containing liquid with an activated charcoal, eluting the streptomycin from the charcoal with an aqueous, water-soluble mineral acid, intimately contacting the eluate with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfomates, and sulfonated castor oil, recovering the organic solvent phase, intimately contacting the organic solvent solution with an aqueous, watersoluble, relatively-strong acid, and recovering the aqueous phase.

11. The method which comprises intimately contacting an aqueous solution of streptomycin with a solution in a substantially water-immiscible organic solvent for soaps of a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil.

12. The method which comprises mixing an aqueous solution of streptomycin with a watersoluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, and then intimately contacting the mixture with a substantially water-immiscible organic solvent for soaps.

13. The method of purifying streptomycin, which comprises intimately contacting an aqueous solution of an impure streptomycin with a substantially water-immiscible organic solvent for soaps and a watersoluble member of the and recovering the polyalkyl-aryl sulfonates, and sulfonated castor oil, recovering the organic solvent phase, washing the organic solvent solution with water, and converting the salt-type derivative of streptomycin therein into a water-soluble salt of streptomycm.

14. The method which comprises intimately contacting an aqueous solution of streptomycin with a substantially water-immiscible organic solvent for soaps and a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, adjusting the pH of the aqueous phase to between about 3.0 and 4.0, separating the organic solvent phase, and converting the salt-type derivative of streptomycin therein into a Water soluble salt of streptomycin.

15. The method which comprises intimately contacting an aqueous solution of streptomycin with a substantially water-immiscible organic solvent for soaps, and an excess of a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, separating the organic solvent phase, and converting the salt-type derivative of streptomycin therein into a water-soluble salt of streptomycin.

WILLIAM A. LOTT. JACK BERNSTEIN. LEON J. HEUSER.

References Cited in the file of this patent UNITED STATES PATENTS 

1. THE METHOD WHICH COMPRISES INTIMATELY CONTACTING AN AQUEOUS SOLUTION OF AN ANTIBIOTIC OF THE GROUP CONSISTING OF STREPTOMYCIN, DIHYDROSTREPTOMYCIN, AND STREPTOTHRICIN WITH A SUBSTANTIALLY WATER-IMMISCIBLE ORGANIC SOLVENT FOR SOAPS AND A WATER-SOLUBLE MEMBER OF THE GROUP CONSISTING OF SULFONATES OF HIGHER FATTY ACID ESTERS, SULFONATES, OF HIGHER FATTY ACID AMIDES, SULFONATES OF PETROLEUM HYDROCARBONS, POLYALKYLARYL SULFONATES, AND SULFONATED CASTER OIL. 